The present invention relates to a method for hydroxylating cholesterol by the action of microorganisms, and more specifically to a method for preparing one or more of either 25-hydroxycholesterol, 17,25-dihydroxycholesterol or 25,26-dihydroxycholesterol from cholesterol. The invention also relates to the aforementioned new dihydroxycholesterols.
For a biological method, in particular, a method for preparing hydroxy-derivatives of steroids including cholesterol by means of microorganisms, a method by which cholesterol is converted into 25-hydroxycholesterol using microorganisms of the genus Streptomyces has been disclosed in Japanese Laid-Open Patent Publication No. 1,23997/95. Also, in the biological conversion, which is interesting, of compounds other than steroids, there are known the methods for preparing 25-hydroxyvitamin D compounds by the hydroxylation of vitamin D compounds using microorganisms, for example, Nocardia autotroihica, Streptomyces roseosporus, Amycolata saturnea, Amycolata autotrophica, Sphingomonas sp. (Japanese Laid-Open Patent Publication Nos. 166090/92, 241197/95).
It is known that cholesterol may be, for example, an intermediate on the chemical synthesis of various kinds of vitamin D compounds (Yuki Gosei Kagaku (Organic Synthetwic Chemistry) 37, 809-829 (1979)), and compounds wherein one or more of the specific sites of cholesterol is/are hydroxylated beforehand may be expected for their use as intermediates on the synthesis of various hydroxylated vitamin D compounds. Indeed, according to the conversion method using microorganisms described in the abovementioned Japanese Laid-Open Patent Publication No. 123997/95, it has been published that cholesterol can be selectively hydroxylated at the 25-position which is preferable in relation to activated vitamin D.
However, the hydroxylation efficiency, according to the method described in said patent publication, is not always satisfactory. From the view point of improving the water-solubility of final compounds derived from cholesterol, for example, vitamin D compounds, it would be also desired to provide not only mono-hydroxylated cholesterols but also further hydroxylated cholesterols, for example, dihydroxylated cholesterols.
The purpose of the present invention is therefore to provide a method for efficiently preparing mono-hydroxylated cholesterols, in particular 25-hydroxycholesterol, and a method for preparing further hydroxylated dihydroxycholesterols, and new dihydroxycholesterols per se.
In the course of intensive study for the accomplishment of the above purpose, the present inventors have found that microorganisms of genera other than the genus Streptomyces described in the abovementioned Japanese Laid-Open Patent Publication No. 123997/95 hydroxylate cholesterol not only at the 25-position but also at the 17- or 26-position.
Thus, the above purpose can be achieved by providing a method, by which cholesterol having formula (I) 
is biologically converted into hydroxylated cholesterols of formula (II) 
(in which R1 is a hydroxyl radical, and R2 and R3 are a hydroxyl radical and a hydrogen atom, respectively, or a hydrogen atom and a hydroxyl radical, respectively), for preparing hydroxylated cholesterols having the formula (II), according to.the invention, comprising
(A) a step in which the aforementioned biological conversion can be carried out, and in which cholesterol having the formula (I) is treated by incubation in the presence of a microorganism, chosen from those that belong to the genus Amycolata and the genus Sphingomonas, or its preparation from cultures and oxygen, and
(B) a step in which at least one of the hydroxylcholesterols having the formula (II) is recovered from the incubation-treated solution.
Among the hydroxylated cholesterols having the formula (II), dihydroxycholesterols of formula (II-b) 
(in which R2 and R3 are a hydroxyl radical and a hydrogen atom, respectively, or a hydrogen atom and a hydroxyl radical, respectively) can be also prepared by another method in which 25-hydroxycholesterol is substituted for cholesterol as a starting material in the above method.
Furthermore, dihydroxycholesterols having the above formula (II-b), that is, 17,25-dihydroxycholesterol and 25,26-dihydroxycholesterol, are compounds that have not been published in literature in the prior art. Therefore, according to the present invention, new compounds, dihydroxycholesterols having the formula (II-b), are also provided.
In the biological conversion according to the present invention, microorganisms and their preparations from cultures can be used, regardless of the kinds of species and strains, provided that they are microorganisms belonging to the genus Amycolata and the genus Sphingomonas and having the capacity to convert cholesterol of the above formula (I) into hydroxylated cholesterols of the formula (II). Mention can be made, as preferred microorganisms, Amycolata saturnea having the abovementioned conversion capacity, in particular, the microorganisms that have been deposited at the National Institute of Bioscience and Human Technology, the Agency of Industrial Science and Technology, the Ministry of International Trade and Industry in Japan with the deposition numbers of FERM BP-5544 (deposited Aug. 7, 1995) and FERM BP-2307 (deposited Feb. 27, 1989), and Amycolata autotrophica, in particular, the strain that has been deposited at American Type Culture Collection in the United States with the deposition number of ATCC 33796 (on deposit since 1996).
Species that belong to the genus Sphingomonas, mali IFO 15500, paucimobilis IFO 13935, parapaucimobilis IFO 15100, vanoikuyae IFO 15102, adhaesiva IFO 15099, capsulata IFO 12533, sanguis IFO 13937, macrogoltabidus IFO 15033 and terrae IFO,15098, although they are inferior in conversion capacity compared to that of microorganisms belonging to the above-described genus Amycolata, can be also used. Here, IFO is a deposition number in the Institute for Fermentation in Japan.
According to the invention, cholesterol (the compound of formula (I)) and/or 25-hydroxycholesterol (the compound of formula (II-a)) being a starting material (or a substrate) will be treated by incubation in the presence of any of said strains or their mycelia from cultures and oxygen. This treatment can be carried out by adding a substrate, at the time of the cultivation of the above strain under the aerobic conditions, into a culture solution, or optionally by adding a substrate into a suspension of, for example, the mycelia as such or the homogenized preparations obtained from cultures of the above strains, followed by incubation with oxygen, for example, with air. The addition of a substrate into a culture solution may be performed either before the cultivation or at a certain period of time after the cultivation. The above mycelia can be prepared by inoculating any of the above strains into a medium containing nutrient sources, followed by aerobic cultivation.
The cultivation of a strain to obtain such bacterial preparation from cultures or the cultivation of a strain carried out with the addition of a substrate can be performed, in principle, in accordance with cultivation methods for general microorganisms, but it is usually preferable to be carried out under aerobic conditions such as shaking liquid culture, aerated and stirred culture, etc.
The media used for the cultivation may be those containing nutrient sources which can be utilized by microorganisms belonging to the genus Amycolata and the genus Sphingomonas, and any of various kinds of synthetic or semi-synthetic media, natural media and the like can be used. For the medium compositions, glucose, maltose, xylose, fructose, sucrose and the like can be used alone or in combination as carbon sources. For nitrogen sources, organic nitrogen sources such as peptone, meat extract, soybean meal, casein, amino acids, yeast extract, urea and the like, and inorganic nitrogen sources such as sodium nitrate, ammonium sulfate and the like can be used alone or in combination. Furthermore, if necessary, for example, salts such as sodium chloride, potassium chloride, calcium carbonate, magnesium sulfate, sodium phosphate, potassium phosphate, cobalt chloride and the like, salts of heavy metals, vitamins can also be used. In case where foaming is furious during the cultivation, various known defoamers can be also added suitably into a medium.
Cultivation conditions can be suitably selected so that said strains can grow well. Usually, the cultivation are performed at pH 6-7.5, at 28-30xc2x0 C. for approximately 2-8 days. Various cultivation conditions described above can be suitably changed depending on the kind and property of a microorganism used, external conditions and the like, and optimized conditions can be easily selected by those skilled in the art.
Alternatively, after completion of the cultivation, bacterial preparation from cultures is prepared by suspending mycelia, which has been separated by centrifugation or filtration, or homogenized mycelia in an appropriate solution. Solutions that can be used for suspending mycelia are the media as described above or buffer solutions such as tris-acetate, tris-hydrochloride, sodium succinate, sodium citrate, sodium phosphate, potassium phosphate and the like and they are used alone or in admixture. For the pH values of the buffer solution, preferably 6.0-9.0 and more preferably 7.0-8.5 can be mentioned.
A substrate can be added into a culture solution or a bacterial suspension in the form of powder or by dissolving in water-soluble organic solvent, for example, ethanol and the like, and the amount to be added, for example, in the case of a culture solution, is preferably 0.15-0.60 mg per 1 ml of the culture solution. When the amount is increased to more than 0.60 mg/ml, conversion rate becomes slow and is not preferable. After the addition of a substrate the substrate can be converted into an objective hydroxylated cholesterol by carrying out the operation of shaking or aeration-agitation and the like at 27-31xc2x0 C. for 1-3 days, preferably approximately for one day, to allow the reaction to proceed under aerobic conditions. In such a conversion reaction, the conversion rate into an objective hydroxylated cholesterol from a substrate can be remarkably increased by adding the substrate and methylated cyclodextrins to a reaction solution.
In a preferred embodiment according to the invention, the incubation treatment described above is therefore carried out further in the presence of methylated cyclodextrins.
Methylated cyclodextrins used according to the invention refer to compounds wherein hydrogen atoms of hydroxyl radicals at the 2-, 3- or 6-position of cyclodextrin are substituted by methyl radicals, and hexakis-(2,6-0-dimethyl)-xcex1-cyclodextrin derived from xcex1-cyclodextrin, heptakis-(2,6-0-dimethyl)-xcex2-cyclodextrin derived from xcex2-cyclodextrin and octakis-(2,6-0-dimethyl)-xcex3-cyclodextrin derived from xcex3-cyclodextrin, which are completely methylated at the 2- and 6-positions, or hexakis-(2,3,6-0-trimethyl)-xcex1-cyclodextrin derived from xcex1-cyclodextrin, heptakis-,(2,3,6-0-trimethyl)-xcex2-cyclodextrin derived from xcex2-cyclodextrin and octakis-(2,3,6-0-trimethyl)-xcex3-cyclodextrin derived from xcex3-cyclodextrin, which are completely methylated at the 2-, 3- and 6-positions, or partially methylated cyclodextrins wherein each of 6, 7 or 8, hydroxyl radicals at the position of the 2-, 3- and 6-positions are partially methylated can be mentioned. In the present invention, one or more of any methylated cyclodextrins is/are selected from those described above and used, but, in particular, partially methylated cyclodextrin derived from xcex2-cyclodextrin is preferably used.
The amount of methylated cyclodextins added is 0.5 mg or more, preferably 0.5-15 mg, and more preferably 1-10 mg per 1 ml of a reaction solution. When the amount of the methylated cyclodextrins added is less than 0.5 mg per 1 ml of a reaction solution, there are sometimes cases where the increase in conversion rate into an objective hydroxylated cholesterol does not become significant compared to that without the addition, and for the amount in the region of 15 mg, foaming takes place in some cases, and it may become necessary to use a defoamer etc. together.
In the method according to the invention, nonionic surfactants may be added into a reaction mixture so as not to decrease the conversion rate described above. Mention can be made, as such a surfactant, of polyoxyethylene.sorbitan fatty acid ester (e.g., Tween(copyright) 80 (Sigma)), sorbitan fatty acid ester (e.g., Span (copyright) 85 (Sigma)), polyoxyethylene ether (e.g., Brij(copyright) 96 (Sigma)) and Triton(copyright) X-100 (Sigma), nonylphenol (e.g., Nonypol(copyright) 45 (Sanyo Chemical Industries, Ltd.), block copolymer of ethylene oxide-propylene oxide (e.g., Pluronic(copyright) L-61 (Asahi Denka Kogyo K.K.), and Dislex(copyright) (Nippon Oil and Fats Co. Ltd.) as an anionic surfactant, Trax (Nippon Oil and Fats Co. Ltd.) and the like.
To isolate an objective hydroxylated cholesterol thus produced from a reaction mixture, various known purification procedures can be selected and carried out in combination. For example, it can be separated and purified by means of adsorption to hydrophobic adsorption resins and elusion, extraction with solvent using ethyl acetate, n-butanol etc., a column chromatography with silica gel etc. or thin layer chromatography, preparative high performance liquid chromatography using a reversed phase column and the like and these can be used alone or suitably in combination, or optionally used repeatedly.
An Amycolata saturnea FERM BP-5544, which is one of the strains being able to be used particularly advantageously in the abovementioned biological conversion, was isolated from soil by the present inventors, and named A-1246 strain, and it is a new strain as described below showing bacteriological properties as follows:
(1) Morphology
Vegetative mycelium develops well on synthetic or natural agar media and branches irregularly. No septum is observed. Spore chains are formed abundantly on glycerin-asparagine agar media, starch-inorganic salts agar media and the like. By microscopic observation, sporulating mycelium branches monopodially with straight spore chains. Usually, the spore chains have three or more spores, and the long spore chains are developed at the late growth phase of the culture with smooth surfaces. The spore is cylindrical in shape and 0.5-0.8xc3x972.5-4.3 xcexcm in size. Sclerotia, sporangia and flagellated spore are not observed.
(2) Growth on Various Media (30xc2x0 C.)
(2-1) Sucrose-nitrate Agar Medium
Growth on the medium is moderate and the color of the reverse side of colonies is pale brown. Aerial mycelium forms moderately and colors creamy. No soluble pigment is produced.
(2-2) Glucose-asparagin Agar Medium
Growth on the medium is slightly poor and the color of the reverse side of colonies is creamy. Aerial mycelium forms moderately and colors white. No soluble pigment is produced.
(2-3) Glycerin-asparagin Agar Medium
Growth on the medium is good and the color of the reverse side of colonies is pale yellow. Aerial mycelium forms well and colors white. No soluble pigment is produced.
(2-4) Starch-inorganic Salts Agar Medium
Growth on the medium is moderate and the color of the reverse side of colonies is creamy. Aerial mycelium forms well and colors white. No soluble pigment is produced.
(2-5) Tyrosine Agar Medium
Growth on the medium is moderate and the color of the reverse side of colonies is reddish brown. Aerial mycelium forms well and colors creamy. Soluble pigment with pale reddish brown color is produced.
(2-6) Nutrient Agar Medium
Growth on the medium is good and the color of the reverse side of colonies is pale yellow. Aerial mycelium forms well and colors white. No soluble pigment is produced.
(2-7) Yeast-malt Extract Agar Medium
Growth on the medium is good and the color of the reverse side of colonies is pale yellow. Aerial mycelium forms slightly poorly and colors white. No soluble pigment is produced.
(2-8) Oatmeal Agar Medium
Growth on the medium is moderate and the color of the reverse side of colonies is creamy. Aerial mycelium forms slightly poorly and colors white. No soluble pigment is produced.
(2-9) Peptone.yeast.iron Agar Medium
Growth on the medium is moderate and the color of the reverse side of colonies is pale brown. Aerial mycelium forms moderately and colors creamy. No soluble pigment is produced.
(3) Physiological Properties
(3-1) Temperature Range for Growth
When nutrient agar medium is used, good growth is observed at the temperature in the range of 20-30xc2x0 C. There is no growth at 10xc2x0 C. or below, and at 40xc2x0 C. or above
(3-2) Distinction Between Aerobic and Anaerobic
Aerobic
(3-3) Liquefaction of Gelatin
positive
(3-3) Hydrolysis of Starch
negative
(3-4) Coagulation and Peptonization of Skim Milk
both negative
(3-5) Formation of Melanin-like Pigment
negative
(3-6) Nitrate Reduction
negative
(4) Utilization of Carbon Sources
When a carbon source is added onto Pridham.Godlieb agar medium and the growth is observed, any carbon sources of the followings: D-glucose, sucrose, D-xylose, inositol, D-mannitol, D-fructose, can be utilized. L-arabinose, L-rhamnose and raffinose cannot be utilized.
(5) Cell Wall Components
As a result of the analysis of cell wall components with whole bacterial lysate, the cell wall of this strain belongs to the type III according to the classification by Lechevalier (International Journal of Systematic Bacteriology, vol. 20, p435-443 (1970)). Mycolic acid is not contained.
It is apparent form the above bacteriological properties that this strain belongs to Actinomycetes, and when these properties were compared with those of known microorganisms reported in the International Journal of Systematic Bacteriology, Vol.36, p29-37 (1986), this strain was almost identical to Amycolata saturnea. As a result of the above, this strain is concluded to belong to Amycolata saturnea, and named an Amycolata saturnea A-1246 strain. After deposited at the National Institute of Bioscience and Human Technology, the Agency of Industrial Science and Technology in Japan as FERM P-15098 on Aug. 7th, 1995, this strain was transferred to the International Depositary Authority in the institute and given the deposition number FERM BP-5544 in compliance with the provisions of the Budapest Convention on International Acknowledgement of the Deposition of Microorganisms for the purpose of Patent Proceedings.
The present invention is illustrated in more detail by the following Examples, which are not intended to limit the invention.
Unless mentioned otherwise, the percentages in the examples below represents percent by weight.